Cloned transmembrane receptor for 24-hydroxylated vitamin d compounds and uses thereof

ABSTRACT

The instant invention relates to the use of 24-hydroxylated vitamin D compounds as therapeutics in mammalian bone fracture repair. In addition, the instant invention relates to novel 24-hydroxylated vitamin D compound receptors which can be employed in the development of compounds capable of facilitating fracture repair in animals. The instant invention also relates to nucleic acids encoding such receptors as well as vectors, host cells, transgenic animals comprising such nucleic acids and screening assays employing such receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.13/306,599, filed Nov. 29, 2011, which is a continuation ofInternational Patent Application No. PCT/US2010/036842, filed Jun. 1,2010, published in English on Dec. 9, 2010 as International PatentPublication No. WO10/141,430, which claims priority to U.S. ProvisionalApplication No. 61/182,951, filed Jun. 1, 2009, all of which areincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Two forms of vitamin D exist in nature: vitamin D₂ (ergocalciferol),which is formed in plants by the UV irradiation of the plant productergosterol, and vitamin D₃ (cholecalciferol), which is formed in animaltissues from near-UV (290-310 nm) irradiation of 7-dehydrocholesterolfound in keratinocytes (123). In animals, vitamin D₃ functions as a keyregulator of mineral ion homeostasis, but first the vitamin must undergotwo modifications in order to be activated. In the liver, vitamin D₃ isinitially hydroxylated at position 25, and in the kidney, it issubsequently hydroxylated at position 1 to produce 1,25-(OH)₂D₃, thehormonal form of vitamin D (1). Upon reaching its target tissues,1,25-(OH)₂D₃ binds to its specific nuclear receptor, the vitamin Dreceptor (VDR), to regulate the transcription of vitamin D target genesresponsible for carrying out physiological actions including: mineralhomeostasis, skeletal homeostasis, and cellular differentiation (2).

The Cyp24a1 gene encodes the CYP24A1 cytochrome P450 enzyme thatcatalyzes the addition of a hydroxyl group on carbon 24 of the vitamin Dsecosteroid backbone. When the substrate is 1,25-(OH)₂D₃, hydroxylationby CYP24A1 leads to the production of 1,24,25-trihydroxyvitamin D₃.1,24,25-trihydroxyvitamin D₃ is the initial reactant in the 24-oxidationpathway that leads to metabolite inactivation (3). Indeed, blockingCYP24A1 cytochrome P450 activity in cell culture systems inhibitscatabolism of, and results in increased accumulation of 1,25-(OH)₂D₃(4). The function of the CYP24A1 protein as an effector of 1,25-(OH)₂D₃breakdown has also been confirmed in vivo. For example, mice deficientfor the Cyp24a1 gene cannot effectively clear 1,25-(OH)₂D₃ from theircirculation (5).

The 25-(OH)D₃ metabolite can also serve as the substrate for the CYP24A1enzyme. Use of 25-(OH)D₃, as the substrate leads to the production of24,25-(OH)₂D₃. Prior to the filing of this application, the potentialbioactivity of 24,25-(OH)₂D₃ remained controversial. For example, theliterature demonstrates that Cyp24a1 is expressed in growth platechondrocytes and that cells from the growth plate respond to24,25-(OH)₂D₃ in a cell maturation-dependent manner (6). However, thegrowth plates from Cyp24a1^(−/−) mice do not show major defects (5).These observations suggested that the absence of CYP24A1 activity doesnot affect growth plate development and that 24,25-(OH)₂D₃ is notrequired for chondrocyte maturation in vivo.

Another aspect of bone biology in which investigators have sought toidentify a role for 24,25-(OH)₂D₃ is fracture repair. Traumatic injuryis a major public health issue. In the United States, close to 10million trauma-induced fractures are reported annually (National Centerfor Health Statistics). United States statistics for the year 2002reported 54 million office visits, 21 million emergency room visits, 4.5million outpatient visits and 2 million hospitalizations dealing withtraumatic injuries. Of the 2 million hospitalizations, 13 millionrelated to bone fractures (United States Bone and Joint Decade web site,www.usbjd.org). Fractures continue to be the leading cause of injuryhospitalization in the United States, accounting for more than one-halfof all injury hospitalizations in 2004-2005 (National Center for HealthStatistics).

With these traumatic fracture statistics in mind, consideration mustalso be given to the increase in the incidence of osteoporotic fracturesthat occurs in individuals after age 65. The aging of the U.S.population will increase the relative impact of musculoskeletalconditions: over the next thirty years, the percent of the populationage 65 and over will increase from 12.8% to 20.0%. Individuals 65 yearsand older, especially women, are more likely to sustain a bone fracture.Each year, roughly 1.5 million people suffer a bone fracture related toosteoporosis (FDA Consumer magazine, January-February 2005 issue).

It has previously been shown that circulating levels of 24,25-(OH)₂D₃increase during fracture repair in chicks due to an increase in renalCYP24A1 activity (7). When the effect of various vitamin D metaboliteson the mechanical properties of healed bones was tested, treatment with1,25-(OH)₂D₃ alone resulted in poor healing (8). However, the strengthof healed bones in chickens fed 24,25-(OH)₂D₃ in combination with1,25-(OH)₂D₃ was equivalent to that measured in a control population fed25-hydroxyvitamin D₃ (8). Such results support a role of 24,25-(OH)₂D₃as an essential vitamin D metabolite for fracture repair in chickens.Furthermore, in light of the signaling pathway associated with1,25-(OH)₂D₃ in chickens, it was postulated that 24,25-(OH)₂D₃ also actsthrough receptor-mediated signaling, and preliminary evidence suggestedthe presence of a non-nuclear membrane receptor for 24,25-(OH)₂D₃ in thechick tibial fracture-healing callus (9,10). Prior to the instantapplication, studies establishing a therapeutic activity for24,25-(OH)₂D₃ in mammalian fracture repair and the molecular nature of a24-hydroxylated vitamin D compound receptor had not been reported.

At present, the only drugs approved for fracture repair/treatment arerecombinant Bone Morphogenetic Proteins (BMPs). Their use is restrictedto anterior lumbar interbody spine fusion, open tibial shaft fractures,and recalcitrant nonunion fractures (14). These treatments are extremelycostly and success rates remain below 70% (15). The pharmaceuticalindustry is working on smaller and cheaper molecules that could activatethe BMP receptors (BMP mimetics), however there have been no publishedresults on such studies.

Although not yet approved for human use, parathyroid hormone (“PTH”)administration has been shown to improve fracture repair in rat studies(16, 17). However, the most dramatic effects come relatively late duringthe repair process. Furthermore, while parathyroid hormone treatment hasfew side effects, it is costly and requires daily injections (18).Accordingly, it is not likely to be a treatment that will be welltolerated by many patients.

Selective prostaglandin receptor agonists have also been considered forstimulation of bone repair. For example, selective agonists for receptorE2 (EP2) and receptor E4 (EP4) have been shown to stimulate fracturerepair in rodents and dogs (19, 20). However, whether these agonists arebeing further developed for clinical use is unknown, as is the potentialthat significant undesired side effects may be associated with theiruse.

It has also recently been shown that bone mass can be regulated from thehypothalamus via the nervous system through adrenergic receptors (25).Based on that finding, it was hypothesized that bone mass may besusceptible to modulation by compounds that block such receptors(“beta-blockers”). For example, propranolol, a common beta-blocker, wasshown to increase bone mass in wild-type mice and repair bone defects inrats (25, 27). Indeed, a large case-control study has suggested thatbeta-blockers reduce the risk of osteoporosis fractures (26). However,whether beta-blockers can be used to improve bone fracture healingwithout eliciting significant side effects has not been determined.

Finally, lipid-lowering drugs, known as statins, have also been shown tostimulate bone formation in vitro and in rodents (21). For example,there has been a report of enhanced fracture repair in mice followingsimvastatin treatment (22). However, the effectiveness of statintreatment for osteoporosis and fracture repair treatment remainscontroversial. The lack of association in randomized trials and theheterogeneity among observational studies do not support an effect ofstatins in preventing fractures (23). This could be due to thepharmacokinetic properties of statins, which are rapidly metabolizedafter one passage through the liver (24). Thus, it remains questionablewhether statins could be used efficaciously for treatment of boneinjuries.

Specifically, there is a need for a therapy that is less costly and moreeasily administrable than the therapies discussed above and that has anacceptable side effect profile.

The instant invention addresses the deficiencies of the compoundscurrently under study by providing new avenues for identifying compoundshaving more desirable traits. Specifically, the instant inventionrelates to novel 24-hydroxylated vitamin D compound receptor which canbe employed in the development of such compounds. In addition, theinstant application provides the first data establishing that24-hydroxylated vitamin D compounds can function as a therapeutic inmammalian fracture repair.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a method ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, the method comprising first contacting a24-hydroxylated vitamin D compound receptor with a candidate compound;and subsequently determining whether the candidate compound binds to the24-hydroxylated vitamin D compound receptor.

In another embodiment, the present invention relates to a method ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, where the 24-hydroxylated vitamin D compound receptorcomprises an amino acid sequence shown in SEQ ID NO: 1 or a sequencehaving at least 90% sequence identity thereto.

In another embodiment, the present invention relates to a method ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, wherein the method involves exposing a cellexpressing a 24-hydroxylated vitamin D compound receptor to thecandidate compound.

In another embodiment, the present invention relates to a method ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, wherein binding is detected by measuring a signaltransduction output that arises downstream of the binding event.

In another embodiment, the present invention relates to methods ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, wherein the binding is detected by measuring theactivation of a member of the ATF family of transcription factors. Inparticular embodiments, the transcription factor activation that ismonitored is ATF4 activation. In alternative embodiments, binding isdetected by measuring activation of a protein kinase capable of directlyor indirectly activating a member of the ATF family of transcriptionfactors. In particular embodiments, the protein kinase activation thatis monitored is protein kinase A (cAMP-dependent protein kinase)activation.

In another embodiment, the present invention relates to a method ofidentifying a compound suitable for enhancing bone fracture repaircomprising, first administering a candidate compound capable of bindingto a 24-hydroxylated vitamin D compound receptor to an animal; andsubsequently determining whether the animal exhibits a change bonefracture repair, as compared with an animal to which the candidatecompound has not been administered and thereby identifying a compoundfor enhancing bone fracture repair.

In another embodiment, the present invention relates to transgenicnon-human mammal, such as, but not limited to, a rodent, comprising adisruption in an endogenous 24-hydroxylated vitamin D compound receptorgene. The present invention also relates to cells and/or tissuesisolated from such a transgenic non-human mammal.

In another embodiment, the present invention relates to an isolatednucleic acid comprising SEQ ID NO: 2. In additional embodiments, thepresent invention relates to vectors, expression vectors, host cells,and transgenic animals comprising the nucleic acid of SEQ ID NO: 2.

In another embodiment, the present invention relates to isolatedpolypeptides comprising the sequence of SEQ ID NO:1, or fragmentsthereof that retain the 24-hydroxylated vitamin D compound bindingactivity of SEQ ID NO: 1, as well as fusion polypeptides comprising suchisolated polypeptides and fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: abnormal fracture repair in Cyp24a1^(−/−) mice. Goldner-stainedsections through the callus from wild-type (A) and mutant (B) mice at 14days post-fracture. The callus is highlighted by a yellow border inwild-type animals. Note the delayed callus formation inCyp24a1-deficient animals.

FIG. 2: rescue of impaired fracture repair in Cyp24a1-deficient mice bytreatment with 24,25-(OH)₂D₃. Osteotomy was performed and animals fromeach genotype were treated daily with vehicle, 1,25-(OH)₂D₃ (67 ng/kg)or 24,25-(OH)₂D₃ (6.7 ug/kg). The fracture callus was harvested at 14days post-osteotomy and bone volume (BV) and tissue volume (TV) weremeasured by histomorphometry using the BioQuant Osteo image analysissoftware. WT, wild-type; cyp24a1−/−, mutant mice deficient for theCyp24a1 gene. “N.S.”=not statistically significant, “*”=p<0.05, and“**”=p<0.01.

FIG. 3A-B: (3A) The amino acid sequence of FAM57B (SEQ ID NO:1). Theone-letter amino acid code is recited. (3B) The nucleotide sequence ofFam57b (SEQ ID NO: 2). Sequence obtained from the listing of Fam57b inthe NCBI's Entrez gene database (http://www.ncbi.nlm.gov/entrez).

FIG. 4. Binding saturation and specificity of the 24-hydroxylatedvitamin D compound receptor. A, B. The full-length 24-hydroxylatedvitamin D compound receptor was subcloned into the pcDNA3.1 expressionvector and expressed by stable transfection into COS7 cells. Membranefractions were prepared by differential centrifugation and bindingassays were performed using [³H]-24,25(OH)₂D₃ in the presence or absenceof an excess of nonradioactive 24,25(OH)₂D₃. Bound and free ligand wereseparated by filtration on glass microfiber filters. Specific bindingwas saturable (A), and can be displaced by an excess of cold24,25(OH)₂D₃, but not by 1,25(OH)₂D₃ or progesterone (B). C, D. COS7cells stably transfected with an expression vector for the24-hydroxylated vitamin D compound receptor were plated in a cellulardielectric spectroscopy apparatus and changes in bioimpedance (dZiec),representative of binding, were measured over a range of compoundconcentrations. C. Response to vitamin D compounds. D. Response tosteroids. The cells showed binding activity specific for 24R,25(OH)₂D₃,the natural epimer of 24,25(OH)₂D.

FIG. 5. Activation of the ATF response element following binding of24,25(OH)₂D to the 24-hydroxylated vitamin D compound receptor. COS7cells stably transfected with an empty expression vector (vector) orwith an expression vector for the 24-hydroxyvitamin D compound receptor(24,25(OH)₂D rep) were transiently transfected with a reporter constructin which the luciferase reporter is under the control of an ATF responseelement. Transfected cells were then treated with ethanol (vehicle) or asaturating dose of 24,25(OH)₂D. A renilla luciferase constitutiveexpression vector was co-transfected to assess efficiency oftransformation. Results (mean±SEM) are expressed as fold-induction overtreatment with vehicle. The response of the 24,25(OH)₂Dreceptor-transfected cells is statistically significant (p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that micedeficient for the 25-hydroxyvitamin D-24-hydroxylase gene, Cyp24a1,exhibit a delay in bone fracture healing. We have discovered that thisdelay can be corrected by exogenous administration of 24,25-(OH)₂D₃,indicating that treatment with vitamin D metabolites hydroxylated atposition 24, such as 24,25-(OH)₂D₃, are useful in the treatment of bonefractures brought about by a trauma or metabolic bone disease such asosteoporosis. Furthermore, the inventors employed innovative techniquesto identify a novel 24,25-(OH)₂D₃ receptor that allows 24,25-(OH)₂D₃ toact in fracture repair via receptor-mediated signaling. This24,25-(OH)₂D₃ receptor can be used to screen for compounds, including24,25-(OH)₂D₃ analogs capable of modulating activity of the receptor.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below.

The term “24-hydroxylated vitamin D compounds”, as used herein, refersto vitamin D hydroxylated at position 24 as well as metabolites andanalogs thereof, including, but not limited to, 24,25-(OH)₂D₃ andanalogs thereof. Metabolites of 24-hydroxylated vitamin D compounds arethose compounds produced via the endogenous metabolism of 24hydroxylated vitamin D. Analogs of 24-hydroxylated vitamin D compoundsrefers to those compounds sharing structural similarity and/orfunctional activity with 24-hydroxylated vitamin D, which may includemetabolites of 24-hydroxylated vitamin D compounds.

The term “24,25-(OH)₂D₃ analogs”, as used herein, refers to thosecompounds sharing structural similarity and/or functional activity with24,25-(OH)₂D₃.

The term “24-hydroxylated vitamin D compound receptors”, as used herein,refers to polypeptide receptors capable of binding 24-hydroxylatedvitamin D compounds, and includes, but is not limited to the24-hydroxylated vitamin D compound receptor having the sequence of SEQID NO: 2 or fragments thereof that retain the ability to bind24-hydroxylated vitamin D compounds.

Use of 24-Hydroxylated Vitamin D Compounds in Mammalian Bone FractureRepair

The present invention involves the first description of a therapeuticactivity for a 24-hydroxylated vitamin D compounds in vivo in mammalianfracture repair. Fracture repair involves response to injury,intramembranous bone formation, chondrogenesis, endochondral boneformation, and bone remodelling. The immediate response to the fracturetrauma results in the infiltration of inflammatory cells, macrophages,and platelets during formation of a hematoma (47). Soon after thefracture event, the bone marrow cells reorganize into regions of highand low cellular density. Within a day of the fracture event, cells inthe high cellular density regions undergo differentiation along theosteoblastic lineage (47). Together with the osteoblasts that line thecortical bone, these differentiating osteoblasts lay down new bone viaan intramembranous pathway to form the ‘hard’ callus of woven boneadjacent to the fracture site. In mice, this takes place as early as 3days post fracture and continues until day 14 post-fracture, withproliferation peaking between days 7-10.

Mesenchymal cells proliferate for several days, and then differentiateinto chondrocytes, leading to the formation of the cartilaginous, ‘soft’callus that bridges the fracture site. Proliferation of these newchondrocytes continues from day 7 to day 21 post-fracture. The softcallus provides the initial stabilization at the fracture site.

The mineralization of the soft callus begins at the interface betweenthe maturing cartilage (hypertrophic chondrocytes) and the newly formedwoven bone of the hard callus. Angiogenesis occurs closely afterhypertrophic chondrocyte mineralization of the matrix, mimickingendochondral bone formation at the growth plate. The hypertrophicchondrocytes undergo apoptosis, and the mineralized cartilage matrix isreplaced by woven bone laid down by the osteoblasts that accompanied theinfiltrating new vascular structures. The new bone repairing thefracture site will be subsequently remodelled by cooperativeosteoblast/osteoclast activity, producing bone that is indistinguishablefrom the original intact bone (48).

Thus fracture healing involves a sequential series of cellular andbiochemical events proceeding from inflammation through intramembranousbone formation, chondrogenesis, endochondral bone formation, and finallyremodeling. Several studies have described a complex pattern of geneexpression that occurs during the course of these events (49-52).Extracellular matrix components are differentially expressed during thedifferent stages of fracture repair. Osteocalcin gene expression isinduced and reaches a maximum around day 15 (47, 48). Collagen type IIand aggrecan are expressed initially but are turned off by 9 days postfracture. This is followed by type X collagen expression when thechondrocytes become hypertrophic (53). The chondrocytes also expressalkaline phosphatase, whose expression peaks around days 17-18 postfracture (54). Taken together, results from gene expression monitoringduring bone repair suggest that the molecular regulation of fracturehealing is complex but mimics some aspects of embryonic skeletalformation (55, 56).

As outlined in Example 1, fracture repair has been compared betweencyp24a1−/− mice and wild-type controls. A delay in the mineralization ofthe cartilaginous matrix of the soft callus in cyp24a1−/− mutant animalshas been measured, which is accompanied by reduced expression ofchondrocyte marker genes. This repair delay and the aberrant pattern ofgene expression is rescued by treatment with 24-hydroxylated vitamin Dcompound, such as 24,25(OH)₂D₃.

The 24-hydroxylated vitamin D compounds of the invention can beformulated into compositions suitable for pharmaceutical administration.The pharmaceutical composition typically includes a 24-hydroxylatedvitamin D compound, such as, but not limited to 24,25-(OH)₂D₃, and apharmaceutically acceptable carrier.

The 24-hydroxylated vitamin D compounds of the invention can beadministered alone or linked to a carrier peptide, such as, for example,a Tat carrier peptide. Other suitable carrier peptides are known andcontemplated, such as the Drosophila Antennapedia homeodomain, where thepeptide is cross-linked via an N-terminal Cys-Cys bond to theAntennapedia carrier (97). Polyarginine is another exemplary carrierpeptide (98, 99).

A pharmaceutical composition of the present disclosure can beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Routes of administration for the24-hydroxylated vitamin D metabolites of this disclosure includeintravenous, intramuscular, intradermal, intraperitoneal, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.

Alternatively, a pharmaceutical composition comprising a 24-hydroxylatedvitamin D composition of this disclosure can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

Pharmaceutical compositions can be administered with medical devicesknown in the art. For example, in a preferred embodiment, a therapeuticcomposition of this disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions disclosed herein is contemplated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example,solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide.Parenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL. (BASF, Parsippany, N.J.), phosphate buffered saline (PBS), ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Inall cases, the composition must be sterile and should be fluid tofacilitate easy syringability. The proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. The composition must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. It maybe preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., the 24-hydroxylated vitamin D compound) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filtersterilizing the resulting solution. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and other required ingredients fromthose enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, one method of preparationis vacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form of atablet, pill, troche, or capsule. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The oral compositions can contain any of the followingingredients (or ones of a similar nature): a binder such asmicrocrystalline cellulose; gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparing such formulations will be apparent to thoseskilled in the art. The polymers can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These may be prepared according to methods known to thoseskilled in the art. For example, liposome formulations can be preparedby dissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of active compound is then introducedinto the container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension. See, for example,U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers to aphysically discrete units suited for unitary dosing of the subject to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationsfor the dosage unit forms of the invention are dictated by, and directlydependent on: (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved; and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals. Here, the therapeutic effect andtreatment relate to bone fracture and ameliorating symptoms related tobone fracture.

Actual dosage levels of the active compound in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active compound which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular active compound, the route ofadministration, the time of administration, the rate of excretion of theactive compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with the activecompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A “therapeutically effective dosage” of a 24-hydroxylated vitamin Dcompound of this disclosure preferably results in a decrease in severityof disease symptoms, an increase in frequency and duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease affliction. For example, for the treatment of bone fracture,a “therapeutically effective dosage” preferably enhances bone fracturehealing by at least about 5%, or more preferably by at least 10%, ormore preferably by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects.

Animal models accepted in the art as models of human bone fracturerepair can be used to test particular 24-hydroxylated vitamin Dcompounds, routes of administration etc., to determine appropriateamounts of the 24-hydroxylated vitamin D compounds of the invention.

The ability of a 24-hydroxylated vitamin D compound to enhance bonefracture repair can be evaluated in an mammalian model system predictiveof efficacy in humans. Alternatively, this property of a composition canbe evaluated by examining the ability of the compound to enhance bonefracture repair via in vitro assays known to the skilled practitionerand described herein. A therapeutically effective amount of atherapeutic compound can enhance bone fracture repair or otherwiseameliorate bone fracture symptoms in a subject.

The 24-Hydroxylated Vitamin D Compound Receptor

The present invention includes the first identification andcharacterization of a cloned polypeptide receptor for 24-hydroxylatedvitamin D compounds, such as 24,25-(OH)₂D₃. Thus, one aspect of thedisclosure pertains to polypeptide receptors capable of binding24-hydroxylated vitamin D compounds. In one embodiment, the polypeptidereceptor has the amino acid sequence included in FIG. 3A as SEQ ID NO.1.

Cyp24a1-deficient mice were used as a source of tissue to clone the24-hydroxylated vitamin D compound receptor. Although such a receptorhad previously been postulated to exist, it had not been identifieddespite significant efforts in the field to do so. The instant inventorsundertook an innovative method to identify the 24-hydroxylated vitamin Dcompound receptor. Specifically, the inventors postulated that, in theabsence of its specific ligand and the loss of a putative negativefeedback loop, the receptor would be overexpressed in the repair callusfrom Cyp24a1^(−/−) animals. Thus, as described in Example 2, below, geneexpression profiling with cDNA microarrays was used to identifystatistically significant overexpression of genes in the callus ofCyp24a1-deficient mice as compared to wild-type mice. Binding analysisof the polypeptides encoded by the overexpressed genes led to theidentification of a polypeptide having the amino acid sequence of SEQ IDNO. 1 as the 24-hydroxylated vitamin D compound polypeptide receptor.

In addition to a polypeptide receptor having an amino acid sequence thatis identical to SEQ ID NO. 1, the invention also encompasses polypeptidereceptors that are “substantially similar” to SEQ ID NO. 1. Suchpolypeptides include those that retain certain structural and functionalfeatures of the polypeptide receptor of SEQ ID NO. 1, yet differ fromthe amino acid sequence of that polypeptide receptor at one or moreamino acid position (i.e., by amino acid substitutions). For example, apolypeptide receptor that is substantially similar to SEQ ID NO. 1 isone that retains the ability to bind 24-hydroxylated vitamin Dcompounds. In certain embodiments, such polypeptides include, but arenot limited to, polypeptides encoded by nucleic acid accession no.NM_(—)029978.1 or NM_(—)031478.4. In additional embodiments, suchpolypeptides include, but are not limited to, polypeptides having theamino acid sequence of accession no. NP_(—)084254.1 or NP_(—)113666.2.One example of a polypeptide that is not substantially identical to SEQID NO. 1 is the polypeptide defined by accession no. NM_(—)001146347.1(mRNA) and NP_(—)001139819.1 (protein), which does not retain bindingactivity. Polypeptides that are variants of the one represented by SEQID NO. 1 can be prepared by substituting amino acid residues within theoriginal SEQ ID NO. 1 polypeptide receptor and selecting polypeptidesthat retain 24-hydroxylated vitamin D compound binding activity. Forexample, amino acid residues of the polypeptide receptor can besystematically substituted with other residues and the substitutedpolypeptides can then be tested in standard assays for evaluating theeffects of such substitutions on the ability of the polypeptide to bind24-hydroxylated vitamin D compounds.

In some embodiments, to retain functional activity, conservative aminoacid substitutions are made. As used herein, the language a“conservative amino acid substitution” is intended to include asubstitution in which the amino acid residue is replaced with an aminoacid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including: basic side chains (e.g., lysine, arginine, histidine); acidicside chains (e.g., aspartic acid, glutamic acid); uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine); nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, praline, phenylalanine, methionine, tryptophan);O-branched side chains (e.g., threonine, valine, isoleucine); andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Other generally preferred substitutions involve replacementof an amino acid residue with another residue having a small side chain,such as alanine or glycine. Amino acid substituted peptides can beprepared by standard techniques, such as automated chemical synthesis.

The effect of the amino acid substitutions on the ability of thepolypeptide to bind 24-hydroxylated vitamin D compounds can be tested instandard assays as well-known in the art and described herein (see, forexample, Example 2).

In one embodiment, a polypeptide of the present invention is at leastabout 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% homologous to the amino acid sequence of thepolypeptide receptor (SEQ ID NO:1), and is capable of binding24-hydroxylated vitamin D compounds.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap that need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples described in the EXAMPLES section of thisdisclosure.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (100), which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch algorithm (101), which hasbeen incorporated into the GAP program in the GCG software package,using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases, for example, to identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (102). BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain homologous amino acidsequences. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (103). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) are useful. See theNational Center for Biotechnology Information (NCBI) website.

Polypeptide receptors of the invention can be prepared by any suitablemethod for polypeptide synthesis, including chemical synthesis andrecombinant DNA technology. Methods for preparing peptides byrecombinant expression in a host cell of DNA encoding the polypeptideare well known in the art (see e.g., Sambrook et al. (104)).

In addition to amino acid-substituted polypeptide receptors, theinvention also encompasses polypeptide receptors having othermodifications relative to the receptor represented by SEQ ID NO: 1. Forexample, the amino-terminus or carboxy-terminus of the peptide can bemodified. The phrase “amino-derivative group” is intended to includeamino-terminal modifications of the polypeptide receptors of theinvention. Examples of such modifications include alkyl, cycloalkyl,aryl, arylalkyl, and acyl groups. A preferred N-terminal modification isacetylation. The N-terminal residue may be linked to a variety ofmoieties other than amino acids such as polyethylene glycols (such astetraethylene glycol carboxylic acid monomethyl ether), pyroglutamicacid, succinoyl, methoxy succinoyl, benzoyl, phenylacetyl, 2-, 3-, or4-pyridylalkanoyl, aroyl, alkanoyl (including acetyl and cycloalkanoyle.g., cyclohexylpropanoyl), arylakanoyl, arylaminocarbonyl,alkylaminocarbonyl, cycloalkyl-aminocarbonyl, alkyloxycarbonyl(carbamate caps), and cycloalkoxycarbonyl, among others.

The phrase “carboxy-derivative group” is intended to includecarboxy-terminal modifications of the polypeptide receptors of theinvention. Examples of such modifications include modification of thecarbonyl carbon of the C-terminal residue to form a carboxyterminalamide or alcohol (i.e., as reduced form). In general, the amidenitrogen, covalently bound to the carbonyl carbon on the C-terminalresidue will have two substitution groups, each of which can be ahydrogen, alkyl or alkylaryl group (substituted or unsubstituted).Preferably the carboxy-derivative group is an amido group, such as—CONH₂, —CONHCH₃, —CONHCH.₂C₆H₅ or —CON(CH₃)₂, but may also be 2-, 3-,or 4-pyridylmethyl, 2-, 3-, or 4-pyridylethyl, carboxylic acid, ether,carbonyl ester, alkyl, arylalkyl, aryl, cyclohexylamide, piperidineamideor other mono or disubstituted amide. Other moieties that can be linkedto the C-terminal residue include piperidine-4-carboxylic acid or amide,and cis- or trans-4-amino-cyclohexa-necarboxylic acid or amide.

Moreover, modification of one or more side chains of non-critical aminoacid residues (e.g., “neutral” residues) may be tolerated withoutaltering the function of the polypeptide receptors. A covalentmodification of an amino acid side chain or terminal residue may beintroduced into the polypeptide receptor by reacting targeted amino acidresidues of the polypeptide receptor with an organic derivative agentthat is capable of reacting with selected side chains or terminalresidues. Examples of typical side chain modifications are describedfurther below.

Other portions of the polypeptide can be derivatized. For example,cysteinyl residues can be reacted with α-haloacetates (and correspondingamines), such as chloroacetic acid or chloroacetamide, to producecarboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues canalso be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloro-mercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. Cysteinyl residues can also reactwith nitric oxide generating three potential derivatives, sulphenic(SOH), sulphinic (SO₂ ⁻) and sulphonic (SO₃ ⁻), with each successivederivative possessing increasing chemical stability. Such derivativescan occur in vivo and can also be synthesized in vitro (105).

Histidyl residues can also be derivatized, e.g., by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Parabromophenacyl bromide is alsouseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

Lysinyl and amino terminal residues can be reacted, for example, withsuccinic or other carboxylic acid anhydrides. Derivatization with theseagents has the effect of reversing the charge of the lysinyl residues.Other suitable reagents for derivatizing α-amino-containing residuesinclude imodoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzedglyoxylate.

Arginyl residues can be modified, e.g., by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed under alkaline conditionsbecause of the high pK_(a) of the guanidine functional group.Furthermore, these reagents can react with lysine as well as arginineepsilon-amino groups.

Tyrosyl residues can be modified, e.g., to incorporate spectral labelsvia reactions with aromatic diazonium compounds or tetranitromethane.Commonly, N-acetylimidizol and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Thetyrosyl residues formed can be labeled with ¹²⁵I or ¹³¹I and used inradioimmunoassays or any other suitable assay.

Carboxyl side groups (aspartyl or glutamyl) can be selectively modified,e.g., by reaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-demethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted, for example, toasparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues can be, e.g., deamidated to formglutamyl and aspartyl residues. In certain embodiments, these residuesare deamidated under mildly acidic conditions. Either form of theseresidues falls within the scope of this invention.

Other modifications can include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (106).

Nucleic Acids Encoding 24-Hydroxylated Vitamin D Compound Receptors

Another aspect of this disclosure pertains to isolated nucleic acidmolecules that encode 24-hydroxylated vitamin D compound receptors ofthis disclosure, portions thereof, as well as complements of thesenucleic acid molecules. An exemplary 24-hydroxylated vitamin D compoundreceptor has the nucleotide sequence identified in FIG. 3B as SEQ ID NO.2.

In other embodiments, the nucleic acid molecule of the invention issufficiently complementary to a nucleotide sequence encoding a24-hydroxylated vitamin D compound receptor of this disclosure such thatit can hybridize under stringent conditions to a nucleotide sequenceencoding a 24-hydroxylated vitamin D compound receptor of thisdisclosure, thereby forming a stable duplex.

In another embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%, or more homologous to a nucleotide sequence encoding a24-hydroxylated vitamin D compound receptor of this disclosure, or aportion, preferably of the same length, of such nucleotide sequence.

The nucleic acids may be present in whole cells, in a cell lysate, or insubstantially pure form. A nucleic acid is “isolated” or rendered“substantially pure” when purified away from other cellular componentsor other contaminants, e.g., other cellular nucleic acids or proteins,by standard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art (see, e.g., 107). A nucleic acid of this disclosure can be,for example, DNA or RNA and may or may not contain intronic sequences.In a preferred embodiment, the nucleic acid is a cDNA molecule.

Recombinant expression vectors which include the nucleic acids of theinvention, and host cells transfected with such vectors, are alsoprovided.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has been linkedand can include a plasmid, cosmid or viral vector. The vector can becapable of autonomous replication or it can integrate into a host DNA.Viral vectors include, e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses. The expression vector can bea yeast expression vector, a vector for expression in insect cells,e.g., a baculovirus expression vector, or a vector suitable forexpression in mammalian cells.

The recombinant expression vectors of the invention can be designed forexpression of the 24-hydroxylated vitamin D compound receptors of theinvention in prokaryotic or eukaryotic cells. For example,24-hydroxylated vitamin D compound receptors of the invention can beexpressed in E. coli, insect cells (e.g., using baculovirus expressionvectors), yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel (108). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

The term “host cell” and “recombinant host cell” are usedinterchangeably herein. Such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications can occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein. A host cell can be any prokaryotic oreukaryotic cell.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a24-hydroxylated vitamin D compound receptor of the invention.Accordingly, the invention further provides methods for producing a24-hydroxylated vitamin D compound receptor of the invention using thehost cells of the invention. In one embodiment, the method includesculturing the host cell of the invention (into which a recombinantexpression vector encoding a 24-hydroxylated vitamin D compound receptorof the invention has been introduced) in a suitable medium such that a24-hydroxylated vitamin D compound receptor of the invention isproduced. In another embodiment, the method further includes isolating a24-hydroxylated vitamin D compound receptor of the invention from themedium or the host cell.

Host cells transformed with nucleotide sequences encoding a24-hydroxylated vitamin D compound receptor may be cultured underconditions suitable for the expression and recovery of the receptor fromcell culture. The protein produced by a transformed cell may be locatedin the cell membrane, secreted or contained intracellularly depending onthe sequence and/or the vector used. As will be understood by those ofskill in the art, expression vectors containing polynucleotides thatencode a 24-hydroxylated vitamin D compound receptor can be designed tocontain signal sequences that direct secretion of a 24-hydroxylatedvitamin D compound receptor through a prokaryotic or eukaryotic cellmembrane. Other constructions may be used to join sequences encoding a24-hydroxylated vitamin D compound receptor to nucleotide sequencesencoding a polypeptide domain that will facilitate purification ofsoluble proteins. Such domains include, but are not limited to: metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.).

The inclusion of cleavable linker sequences, such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.), between thepurification domain and the 24-hydroxylated vitamin D compound receptorencoding sequence may be used to facilitate purification. One suitableconstruct includes a nucleic acid encoding a 24-hydroxylated vitamin Dcompound receptor and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on immobilized metal ion affinitychromatography (IMIAC; described in Porath, J. et al. (109)), while theenterokinase cleavage site provides a means for purifying the24,25-(OH)₂D₃ polypeptide receptor from the fusion protein. Althoughdiscussed above with reference to facilitating purification, the instantinvention embraces alternative uses for fusion proteins comprising a24-hydroxylated vitamin D compound receptor fused to another polypeptidesequence, such as for labeling or cellular signaling. A discussion ofvectors that express fusion proteins is provided in Kroll, D. J. et al.(110).

Also within the invention are nucleic acids encoding fusion proteins inwhich a portion of a 24-hydroxylated vitamin D compound receptorpolypeptide is fused to an heterologous polypeptide (e.g., a markerpolypeptide or a fusion partner) to create a fusion protein. Theinvention also includes, for example, isolated polypeptides (and thenucleic acids that encode these polypeptides) that include a firstportion and a second portion; the first portion includes, e.g., a24-hydroxylated vitamin D compound receptor polypeptide, and the secondportion includes an immunoglobulin constant (Fc) region or a detectablemarker, wherein the detectable marker can be, but is not limited to,β-galactosidase, invertase, green fluorescent protein, luciferase,chloramphenicol, acetyltransferase, beta-glucuronidase, exo-glucanase orglucoamylase.

Transgenic Animals Relating to 24-Hydroxylated Vitamin D CompoundReceptors

The present document further encompasses transgenic animals capable ofexpressing natural or recombinant 24-hydroxylated vitamin D compoundreceptors at elevated or reduced levels compared to the normalexpression level. Also included are transgenic animals (“24-hydroxylatedvitamin D compound receptor knockout”) which do not express functional24-hydroxylated vitamin D compound receptor as a result of one or moreloss of function mutations, including a deletion, of the 24-hydroxylatedvitamin D compound receptor gene. Preferably, such a transgenic animalis a non-human mammal, such as a pig, a sheep or a rodent. Mostpreferably the transgenic animal is a mouse or a rat. Such transgenicanimals may be used in screening procedures to identify agonists and/orantagonists of 24-hydroxylated vitamin D compound receptor activity, aswell as to test for their efficacy as treatments for diseases in vivo.

Detailed methods for generating non-human transgenic animals aredescribed in further detail below and in Example 4. Transgenic geneconstructs can be introduced into the germ line of an animal to make atransgenic animal. For example, one or several copies of the constructmay be incorporated into the genome of a mammalian embryo by standardtransgenic techniques.

In additional exemplary embodiments, transgenic non-human animals areproduced by introducing transgenes encoding a 24-hydroxylated vitamin Dcompound receptor into the germline of the non-human animal. Embryonaltarget cells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonal target cell. The specific line(s) of anyanimal used are selected for general good health, good embryo yields,good pronuclear visibility in the embryo, and good reproductive fitness.In addition, the haplotype is a significant factor.

Introduction of the transgene into the embryo can be accomplished by anymeans known in the art such as, for example, microinjection,electroporation, or lipofection. For example, but not by way oflimitation, a 24-hydroxylated vitamin D compound receptor transgene canbe introduced into an mammal by microinjection of the construct into thepronuclei of the fertilized mammalian egg(s), causing one or more copiesof the construct to be retained in the cells of the developingmammal(s). Following introduction of the transgene construct into thefertilized egg, the egg may be incubated in vitro for varying amounts oftime, or reimplanted into the surrogate host, or both. In vitroincubation to maturity is included. One common method in to incubate theembryos in vitro for about 1-7 days, depending on the species, and thenreimplant them into the surrogate host.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the construct by Southern blot analysis of the segmentof tissue. If one or more copies of the exogenous cloned constructremains stably integrated into the genome of such transgenic embryos, itis possible to establish permanent transgenic animal lines, such as themammals detailed above, carrying the transgenically added construct.

Litters of transgenically altered animals can be assayed after birth forthe incorporation of the construct into the genome of the offspring.Preferably, this assay is accomplished by hybridizing a probecorresponding to the DNA sequence coding for the desired recombinantprotein product or a segment thereof onto chromosomal material from theprogeny. Those progeny found to contain at least one copy of theconstruct in their genome are grown to maturity.

For the purposes of this document a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from one or more gametes. Thus, the gamete nuclei must beones that are naturally compatible, i.e., ones that result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to biological considerations, physical ones also govern theamount (e.g., volume) of exogenous genetic material that can be added tothe nucleus of the zygote or to the genetic material that forms a partof the zygote nucleus. If no genetic material is removed, then theamount of exogenous genetic material that can be added is limited by theamount that will be absorbed without being physically disruptive.Generally, the volume of exogenous genetic material inserted should notexceed about 10 picoliters. The physical effects of addition must not beso great as to physically destroy the viability of the zygote. Thebiological limit of the number and variety of DNA sequences that can beintroduced will vary depending upon the particular zygote and functionsof the exogenous genetic material and will be readily apparent to oneskilled in the art. This is because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

The number of copies of the transgene constructs that are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount that enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct are generated to insure that one copy is functional.There will often be an advantage to having more than one functioningcopy of each of the inserted exogenous DNA sequences to enhance thephenotypic expression of the exogenous DNA sequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and used in theart.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and/or expression, although any tissues or celltypes may be used for this analysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzymatic and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout. Where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. When in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using these methods, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the presentdescription will include exogenous genetic material. As set out above,the exogenous genetic material will, in certain embodiments, be a DNAsequence that results in the production of a 24-hydroxylated vitamin Dcompound receptor. Further, in such embodiments the sequence will beattached to a transcriptional control element, e.g., a promoter, whichpreferably allows the expression of the transgene product in a specifictype of cell.

Blastocytes offer a second type of target cell for transgeneintroduction into a non-human animal. When a developing non-human embryois cultured in vitro to the blastocyst stage, it can be targeted forretroviral infection (111). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1986). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (112, 113). Transfection is easily andefficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (113, 114). Alternatively, infection can beperformed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (115). Most of the founders will be mosaicfor the transgene since incorporation occurs only in a subset of thecells that formed the transgenic non-human animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(115).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (116-119). Transgenes can beefficiently introduced into the ES cells by DNA transfection or byretrovirus-mediated transduction. Such transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells thereafter colonize the embryo and contribute to the germ line ofthe resulting chimeric animal (120).

Also provided are non-human transgenic animals, where the transgenicanimal is characterized by having an altered 24-hydroxylated vitamin Dcompound receptor gene, preferably as described above, as models for24-hydroxylated vitamin D compound receptor function. Alterations to thegene can include deletions or mutations that result in a loss of genefunction; or the introduction of an exogenous gene, such as one having anucleotide sequence with targeted or random mutations, or from anotherspecies; or a combination of the foregoing. The transgenic animals maybe either homozygous or heterozygous for the alteration. As described indetail below, such animals and cells derived therefrom are useful forscreening biologically active agents that may modulate 24-hydroxylatedvitamin D compound receptor function. The screening methods are ofparticular use for determining the specificity and action of potentialtherapies for bone fracture repair.

Another aspect pertains to a transgenic nonhuman animal having afunctionally disrupted endogenous 24-hydroxylated vitamin D compoundreceptor gene but carrying within in its genome, and expressing, atransgene encoding a heterologous 24-hydroxylated vitamin D compoundreceptor (e.g., a 24-hydroxylated vitamin D compound receptor fromanother species). Preferably, the animal is a mouse and the heterologous24-hydroxylated vitamin D compound receptor is a human 24-hydroxylatedvitamin D compound receptor. Animals, or cell lines derived from such ananimal, which has been reconstituted with human 24-hydroxylated vitaminD compound receptor, can be used to identify agents that inhibit human24-hydroxylated vitamin D compound receptor in vivo and in vitro. Forexample, a stimulus that induces signaling through human 24-hydroxylatedvitamin D compound receptor can be administered to the animal, or cellline, in the presence and absence of an agent to be tested and theresponse in the animal, or cell line, can be measured. An agent thatinhibits human 24-hydroxylated vitamin D compound receptor in vivo or invitro can be identified based upon a decreased response in the presenceof the agent compared to the response in the absence of the agent.

Methods of Screening Employing the 24-Hydroxylated Vitamin D CompoundReceptor

The characterization of a transmembrane receptor for a 24-hydroxylatedvitamin D compound provided herein identifies a novel target forpharmacological intervention in bone fracture repair. The clonedreceptor can be used to screen for compounds, such as, but not limitedto, vitamin D analogs having increased binding activity for thereceptor.

The 24-hydroxylated vitamin D compound receptor, whether natural orrecombinant, may be employed in a screening process for compounds thatbind the receptor and that activate (agonists) or inhibit activation(antagonists) of the 24-hydroxylated vitamin D compound receptor. Thus,24-hydroxylated vitamin D compound receptors may also be used to assessthe binding of small molecule substrates and ligands found in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics (121). Inaddition, a number of vitamin D analogs have been synthesized (28). Thusan extensive libraries of compounds are readily available for screening.

Rational design of candidate compounds likely to be able to interactwith a 24-hydroxylated vitamin D compound receptor may be based uponstructural studies of the molecular shapes of the 24-hydroxylatedvitamin D compound receptor. One means for determining which sitesinteract with specific other sites is a physical structuredetermination, e.g., X-ray crystallography or two-dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) (122).

An alternative to rational design uses a screening procedure thatinvolves producing appropriate cells that express the 24-hydroxylatedvitamin D compound receptor on the surface thereof. Such cells includecells from animals, yeast, Drosophila or E. coli. Cells expressing thereceptor (or cell membrane containing the expressed receptor) are thencontacted with a test compound to observe binding, or stimulation orinhibition of a functional response. For example, Xenopus oocytes may beinjected with 24-hydroxylated vitamin D compound receptor mRNA orpolypeptide, and currents induced by exposure to test compounds can bemeasured by use of voltage clamps measured.

Ligand-receptor interactions generally trigger signal transductioncascades that translate binding into an intracellular response toregulate cellular events such as proliferation, differentiation,secretion, or apoptosis. The propagation and amplification of thebinding signal involve a wide array of specialized enzymes, such asprotein kinases, and often culminate in the regulation of genetranscription through specific transcription factors. Thus, theidentification of the cascade relevant to the 24-hydroxylated vitamin Dcompound receptor, as outlined in Example 3, allows for alternativescreening assay, such as, but not limited to, those based onproliferation and/or differentiation. For example, microphysiometricassays may be employed to assay 24-hydroxylated vitamin D compoundreceptor activity. Activation of a wide variety of secondary messengersystems by ligand binding to membrane receptors results in extrusion ofsmall amounts of acid from a cell. The acid formed is largely the resultof the increased metabolic activity required to fuel the intracellularsignaling process. The pH changes in the media surrounding the cell arevery small but are detectable by, for example, the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of a receptorthat is coupled to an energy utilizing intracellular signaling pathway.

In certain embodiments of the present invention, cellular dielectricspectroscopy is used to ascertain binding specificity (124). Thetechnology is based on applying electrical current to cells within amicroplate format and measuring changes in impedance. For example, butnot by way of limitation, the CellKey System (MDS AnalyticalTechnologies, Concord, ON, Canada) can be used in the context of theinstant invention. The CellKey System is an impedance-based, label-freetechnology available in 96-well format that measures changes in theimpedance (dZ) of a cell layer that occur in response to receptorstimulation. In certain embodiments, COS-7 cells stably transfected withthe 24-hydroxylated vitamin D compound receptor are seeded at 150,000cells per well in 150 μl of growth medium (high-glucose DMEM with HEPES,10% FBS, and 400 μg/ml of Geneticin). The following day, cells arewashed with HBSS buffer (Hank's balanced salt solution containing 20 mMHEPES, pH 7.4, and 0.1% BSA), then equilibrated for 1 to 2 hours in 150μl of HBSS. The plate is installed onto the system to obtain a baselinereading. Compounds (in 15 μl of HBSS) are added to all wellssimultaneously and the instrument actively measures the impedance ineach well. Measurements are carried for 15 minutes after compoundaddition to monitor cellular responses.

In certain embodiments of the present invention, the commercial CignalFinder reporter system from SABiosciences (Frederick, Md.) is used toidentify the signal transduction pathway acting downstream of the24-hydroxylated vitamin D compound ligand—24-hydroxylated vitamin Dcompound receptor interaction. In particular, non-limiting, embodiments,this system consists of sets of vectors that each contain a distinctcis-acting enhancer element upstream of the luciferase reporter gene.These vectors are transiently transfected into suitable cells. A givenstimulus, such as the binding of a 24-hydroxylated vitamin D compound toits receptor, initiates a signal transduction cascade that ultimatelyresults in the binding of a specific transcription factor to itsresponse element. This in turn leads to increased expression of thereporter gene, providing a convenient readout.

In certain embodiments of the present invention where the Cignal Finderreporter system is employed, COS-7 cells stably transfected with the24-hydroxylated vitamin D compound receptor are seeded in 24-well tissueculture plates (Nunc, Roskilde, Denmark) at a density of 20,000 cellsper well in growth medium (high-glucose DMEM with HEPES, 10% FBS, and400 μg/ml of Geneticin). The next day the medium is changed to 0.5 mlgrowth medium without selection antibiotic. The cells are thentransfected with the Cignal AARE Reporter vector (SABiosciences,Frederick, Md., USA): 250 ng of DNA is mixed with 50 μl of OptiMEM(Invitrogen, Grand Island, N.Y., USA) medium and 1.6 μl of Surefecttransfection reagent is mixed with 50 μl of OptiMEM. After a five minuteincubation period, the DNA and transfection reagent are mixed, furtherincubated for twenty minutes, and the mixture is deposited on top of thecells. After 24 hours, the medium is changed to serum-free DMEM, and thecells are starved overnight (16-18 h). Then, different concentrations of24-hydroxylated vitamin D compound are added to the cells. Followingincubation, cells are washed with PBS and lysed with lysis buffer. Theactivity of firefly and renilla luciferases are measured sequentially ona Sirius Luminometer (Berthold Detection Systems GmbH, Pforzheim,Germany). The specific luciferase activity is expressed as the ratio offirefly/renilla luciferase activity. The specific luciferase activity ofeach treatment group is normalized to the specific activity of acontrol, vehicle-treated group. These values are shown in the graphs asrelative luciferase activity.

In certain embodiments, it is useful to confirm the binding of thetranscription factor to its cognate response element uponligand-receptor interaction using a separate assay. For example, but notby way of limitation, oligonucleotides corresponding to the binding siteof the transcription factor can be synthesized and tested for bindingusing nuclear extracts in Electrophoretic Mobility Shift Assays (EMSAs).In particular embodiments of such assays, nuclear extracts are preparedas described previously (73) using the technique of Andrews and Faller(75). Labeled probe is then added to the binding reaction mixture andthe binding reactions are size-fractionated on non-denaturing 6%polyacrylamide gels. The gel is then dried and autoradiographed. Bindingof the transcription factor to the probe is induced by 24-hydroxylatedvitamin D compound treatment of the cells and result in a complex withreduced electrophoretic mobility (73, 75-70).

In certain embodiments of the present invention, Promega's SignaTECTprotein kinase assay systems (Promega Corporation, Madison, Wis.) areused to characterize specific protein kinase pathways operatingdownstream of the 24-hydroxylated vitamin D compoundligand—24-hydroxylated vitamin D compound receptor interaction. Althoughcommonly used kinase systems can also be employed in the context of theinstant invention, the SignaTECT system overcomes the drawbacks ofcommonly used kinase assay methods that rely on the capture ofphosphorylated peptide substrates on phosphocellulose (80). TheSignaTECT assay is straightforward and requires phosphorylation andbinding of the biotinylated substrate to a biotin capture membrane.Unincorporated [γ-32P]ATP is removed by a simple wash procedure. Washingalso removes nonbiotinylated proteins that have been phosphorylated byother kinases in the sample. The bound, labeled substrate is quantitatedby scintillation counting, phosphorimaging analysis or by usingautoradiography. SignaTECT Protein Kinase Assay Systems are availablefor cAMP-Dependent protein kinase, protein kinase C,calcium/calmodulin-dependent protein kinase II (CaM KII), DNA-dependentprotein kinase, tyrosine kinases, and cdc2 protein kinase.

In certain embodiments, the present invention relates to methods ofidentifying a compound capable of binding to a 24-hydroxylated vitamin Dcompound receptor, wherein the binding is detected by measuringactivation of a member of the ATF family of transcription factors. Inparticular embodiments, the transcription factor activation that ismonitored is ATF4 activation. In alternative embodiments, binding isdetected by measuring activation of a protein kinase capable of directlyor indirectly activating a member of the ATF family of transcriptionfactors. In particular embodiments, the protein kinase activation thatis monitored is protein kinase A (cAMP-dependent protein kinase)activation.

Still another approach is to use solubilized, unpurified or solubilized,purified polypeptide or peptides, for example extracted from transformedeukaryotic or prokaryotic host cells. This allows for a “molecular”binding assay with the advantages of increased specificity, the abilityto automate, and high drug test throughput.

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. A knownligand for a receptor, when in purified form, can be radiolabeled tohigh specific activity (50-2000 Ci/mmol) for binding studies. Adetermination is then made that the process of radiolabeling does notdiminish the activity of the ligand towards its receptor. Assayconditions for buffers, ions, pH and other modulators such asnucleotides are optimized to establish a workable signal to noise ratiofor both membrane and whole cell receptor sources. For these assays,specific receptor binding is defined as total associated radioactivityminus the radioactivity measured in the presence of an excess ofunlabeled competing ligand. Where possible, more than one competingligand is used to define residual nonspecific binding.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfacesInhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed.

Tissues derived from 24-hydroxylated vitamin D compound receptorknockout animals may be used in receptor binding assays to determinewhether the potential drug (a candidate ligand or compound) binds to the24-hydroxylated vitamin D compound receptor. Such assays can beconducted by obtaining a first receptor preparation from the transgenicanimal engineered to be deficient in 24-hydroxylated vitamin D compoundreceptor production and a second receptor preparation from a sourceknown to bind any identified 24-hydroxylated vitamin D compound receptorligands or compounds. In general, the first and second receptorpreparations will be similar in all respects except for the source fromwhich they are obtained. For example, if brain tissue from a transgenicanimal (such as described above and below) is used in an assay,comparable brain tissue from a normal (wild type) animal is used as thesource of the second receptor preparation. Each of the receptorpreparations is incubated with a ligand known to bind to 24-hydroxylatedvitamin D compound receptors, both alone and in the presence of thecandidate ligand or compound. Preferably, the candidate ligand orcompound will be examined at several different concentrations.

The extent to which binding by the known ligand is displaced by the testcompound is determined for both the first and second receptorpreparations. Tissues derived from transgenic animals may be used inassays directly or the tissues may be processed to isolate membranes ormembrane proteins which are themselves used in the assays. A preferredtransgenic animal is the mouse. The ligand may be labeled using anymeans compatible with binding assays. This would include, withoutlimitation, radioactive, enzymatic, fluorescent or chemiluminescentlabeling.

Furthermore, antagonists of 24-hydroxylated vitamin D compound receptoractivity may be identified by administering candidate compounds, etc, towild type animals expressing functional 24-hydroxylated vitamin Dcompound receptor, and animals identified which exhibit any of thephenotypic characteristics associated with reduced or abolishedexpression of 24-hydroxylated vitamin D compound receptor function.

EXAMPLES

The present invention will be better understood by reference to thefollowing Examples, which are provided as exemplary of the invention,and not by way of limitation.

Example 1 Role of 24-Hydroxylated Vitamin D Compounds in MammalianFracture Repair

A Cyp24a1-deficient mouse strain was used to determine the role of24-hydroxylated vitamin D compounds during mammalian fracture repair. Inwild-type mice, there is a significant increase in local expression ofCyp24a1 mRNA in the tibiae subjected to an osteotomy as compared to theunfractured contralateral tibiae. To identify the role of this change ingene expression on callus formation, four-month-old wild-type andCyp24a1-deficient mice were subjected to a stabilized, transversemid-diaphysial fracture of the tibia. Bones were collected at days 14and 21 post-fracture and analyzed for histology and gene expression.Examination of the callus sections stained by the Goldner method showedthat the homozygous mutant animals had delayed callus formation whencompared to wild-type littermates (FIG. 1).

Rescue of the impaired fracture healing in Cyp24a1-deficient mice bysubcutaneous injection of 24,25-(OH)₂D₃ (6.7 μg/kg) or 1α,25-(OH)₂D₃ (67ng/kg) was attempted. Control groups were injected with the vehicle(propylene glycol). Treatment with 1α,25-(OH)₂D₃ had no effect onfracture repair. Daily injection with 24,25-(OH)₂D₃ normalized thehistological appearance of the callus and the measured statichistomorphometric index (BV/TV, FIG. 2). The treatment with24,25-(OH)₂D₃ also rescued and normalized type X collagen mRNAexpression at all time points studied. These results indicate that24-hydroxylated vitamin D compounds play an important role in themechanisms leading to normal fracture healing.

Example 2 Isolation of a 24-Hydroxylated Vitamin D Compound Receptor

Cyp24a1-deficient mice were used as a source of tissue to clone a24-hydroxylated vitamin D compound receptor. Although such a receptorhad previously been postulated to exist, it had not yet been identified,despite significant efforts in the field to do so. The instant inventorsundertook an innovative method to identify a 24-hydroxylated vitamin Dcompound receptor. Specifically, the inventors postulated that in theabsence of its specific ligand and the loss of a putative negativefeedback loop, the receptor would be overexpressed in the repair callusfrom Cyp24a1^(−/−) animals. Thus gene expression profiling with cDNAmicroarrays was used to identify statistically significantoverexpression of genes in the callus of Cyp24a1-deficient mice ascompared to wild-type mice. RNA was extracted from the repair callus ofthree control (Cyp24a1^(+/−)) and three mutant (Cyp24a1^(−/−)) mice at14 days post-osteotomy (a time point where significant differences inthe expression of differentiation markers has been measured usingRT-qPCR). This led to the identification of a restricted set of genes(Table 1). Table 1 provides a summary of gene expression monitoring bycDNA microarrays in fracture callus from wild-type or cyp24a1-deficientmice.

TABLE 1 Gene F.C. Gene Title ID Function (KO/WT) Small proline richSprr2a involved in epithelial different., increased in 5.3 proteinfamily allergic reaction in bronchi BC057627 metal binding, nucleic acidbinding 5.2 Chemokine ligand 1 Cxcl1 Angiogenic chemokine (mousehomologue of IL- 2.59 8) 1500002O20Rik no described function 2.39Tenascin N/maybe W Tnn W inhibits preOBs prolifer. & different. during2.37 endoch.oss., increased in fracture repair 2310046K23Rikhypothetical protein of no described function 2.35 1500016O10Rik nodescribed function, Integral to membrane 2.23 protein 1110020A10Rik nodescribed function 2 5730419F03Rik no described function, expressed inmouse skin 0.49 Histone deacytelase 4 HDAC4 regulates chondrocytehypertrophy &endoch. bone 0.48 formation by inh.of RunX2 2310009E04RikCarbohydrate kinase 0.452 2310009E04Rik no described function 0.45Mm.196290 Oligonucleotide/Oligosachharide-binding fold 0.44 containingprotein U46068 no known function 0.42 Ectodysplasin A2 Eda2r involved inhair, sweat gland and teeth loss in 0.412 isoform receptor humans andmice SH3 domain protein SH3d19 no described function, expressed in mouseskin 0.38 D19 mouse ATPase p5 ATP13a3 ATPase activity in all tissues0.38 member Rufy1 RUN and Rrad lipid, metal, protein binding-involved in0.27 FYVE domain 1 endocytosis $ protein transport & cell migrationKeratin 8 Krt8 Intermediate filament protein involved in 0.18 epithelialcytoskeletal organization similar to keratin, LOC434261 no describedfunction 0.16 cytokeratin 8 similar to keratin, LOC675884 no describedfunction 0.157 cytokeratin 8 Keratin 18 Krt18 Intermediate filamentprotein involved in 0.15 epithelial cytoskeletal organization TGF-beta 1induced TSC22 involved in ocular, maxilla, mandible, skull, and 0.1transcript 4 facial gland developmentStatistical analysis by t test showing significant changes inexpression. Gene highlighted in red were initially selected for furtheranalysis. F.C., fold change; KO/WT, knock-out (cyp24a1-deficient) overwild-type ratio.

Genes highlighted in bold in Table 1 were further characterized sincethey were found to be overexpressed in Cyp24a1^(−/−) callus and were ofpreviously unknown function. Full-length cDNAs for these selectedtargets were subcloned into an expression vector and expressed bytransient transfection into COS-7 cells. Membrane fractions wereprepared by differential centrifugation and binding assays wereperformed using [³H]-24,25-(OH)₂D₃ in the presence or absence of a200-fold excess of nonradioactive 24,25-(OH)₂D₃. Bound and free ligandwere separated by filtration on glass microfiber filters. Specificbinding (total binding minus binding in the presence of excessnonradioactive ligand) measured in membrane fractions from cellstransfected with a given cDNA was considered evidence that a given cDNAencodes a receptor for 24,25-(OH)₂D₃.

Clone 1500016O10Rik (also named Fam57b in the Entrez Gene database) is a1892 bp cDNA annotated in databases as encoding a hypotheticaltransmembrane protein whose predicted amino acid sequence is listed inFIG. 3. The data show that FAM57B expressed in COS-7 cells binds[³H]-24R,25-(OH)2D3 in a specific and saturable manner (FIG. 4). Nospecific binding was measured when the cells were transfected with theempty vector or with expression vectors for the other clones highlightedin Table 1. These results show that Fam57b encodes a transmembranereceptor for 24-hydroxylated vitamin D compounds.

Example 3 Characterization of a 24-Hydroxylated Vitamin D CompoundReceptor

The initial step in characterizing the binding activity of a24-hydroxylated vitamin D compound receptor involves stably transfecting24-hydroxylated vitamin D compound receptor cDNA into COS-7 cells.Membrane fractions of transfected COS-7 cells are prepared bydifferential centrifugation (66): cells are homogenized in buffer A (25mM HEPES, 10 mM NaCl, 1 mM DTT at pH 7.4) and centrifuged at 20,000 gfor 10 minutes. The resulting supernatant is then re-centrifuged at20,000 g for 2×30 minutes. The resulting pellet is resuspended in bufferB (25 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1 mM DTT at pH 7.4) and is usedas a membrane fraction for binding assays.

Binding assays are performed on ice for 60 min in buffer B using[³H]-24R,25(OH)₂D₃ (50 Ci/mmol; Amersham) in the presence or absence ofa 200-fold excess of nonradioactive 24,25(OH)₂D₃. Bound and free ligandare separated by filtration on glass microfiber filters soaked in bufferB. The filters are rinsed with 10 ml of buffer B and are counted forradioactivity using a scintillation counter (36, 37, 66). Specificbinding is then calculated as total binding minus non-specific bindingmeasured in the presence of the excess of nonradioactive ligand.

Saturation binding analysis is performed using 100 μg of membranefraction and 0.1 to 5 nM of [³H]-24R,25(OH)₂D₃ and plotting specificbinding as a function of ligand concentration (FIG. 4A). Saturationbinding is repeated several times to minimize intra-assay variation andcalculate binding affinity with accuracy. Affinity is determined usingthe saturation binding algorithm of the Prism software (GraphPadSoftware Inc., LaJolla, Calif.).

Binding specificity is further refined by performing competition bindingassays on the membrane fractions with various nonradioactive vitamin Dmetabolites and other steroids (FIG. 4B). Binding is performed using 1nM of [³H]-24R,25(OH)₂D₃ and 100 μg of membrane fraction. Competebinding is done using 10-200 fold excess of 24,25(OH)₂D₃ (controldisplacement curve). Compete binding is also done using 10-200 foldexcess of 25(OH)D₃, 24S,25(OH)₂D₃ (the non-natural epimer of24,25(OH)₂D₃), 1α(OH)D₂, 1α,24(OH)D₂, 1,24,25(OH)₃D₃, as well asdexamethasone, estradiol, and testosterone. These experiments are toconfirm the specificity of the 24-hydroxylated vitamin D compoundreceptor for 24,25(OH)₂D₃ and identify vitamin D metaboliteshydroxylated at position 24 that could be higher affinity ligands forthe receptor.

Another method used to ascertain binding specificity is cellulardielectric spectroscopy (124). The technology is based on applyingelectrical current to cells within a microplate format and measuringchanges in impedance. The CellKey System (MDS Analytical Technologies,Concord, ON, Canada) is an impedance-based, label-free technologyavailable in 96-well format that measures changes in the impedance (dZ)of a cell layer that occur in response to receptor stimulation. COS-7cells stably transfected with the 24-hydroxylated vitamin D compoundreceptor are seeded at 150,000 cells per well in 150 μl of growth medium(high-glucose DMEM with HEPES, 10% FBS, and 400 μg/ml of Geneticin). Thefollowing day, cells are washed with HBSS buffer (Hank's balanced saltsolution containing 20 mM HEPES, pH 7.4, and 0.1% BSA), thenequilibrated for 1 to 2 hours in 150 μl of HBSS. The plate is installedonto the system to obtain a baseline reading. Compounds (in 15 μl ofHBSS) are added to all wells simultaneously and the instrument activelymeasures the impedance in each well. Measurements are carried for 15minutes after compound addition to monitor cellular responses. Usingthis technology, it is seen that the recombinant receptor specificallybinds 24,25(OH)₂D, with no cross-reactivity to the other vitamin Dmetabolites that we have tested (FIG. 4A) or to other steroid hormones(FIG. 4B).

A combination of Northern blot assays, TaqMan assays, in situhybridization, and immunochemistry are used to assess the expressionpattern of the 24-hydroxylated vitamin D compound receptor duringdevelopment and in adult tissues. In a first experiment, a commercialNorthern blot containing poly A⁺ RNA from mouse embryos (7-day, 11-day,15-day, and 17-day; BD Biosciences Canada, Mississauga, ON) are probedwith a 24-hydroxylated vitamin D compound receptor probe to determinedevelopmental onset of expression of the RNA.

Embryos are then collected at intervals from the time of onset ofexpression as determined above. The fixed embryos are embedded inparaffin and sectioned for in situ hybridization with a 24-hydroxylatedvitamin D compound receptor riboprobe. Briefly, sections are dewaxed inxylene, rehydrated in serial ethanol dilutions, and re-fixed in 4%paraformaldehyde (PFA) in PBS. This is followed by proteinase Ktreatment, short PFA fixation, and blocking with 0.1 Mtriethanolamine/acetic anhydride. The treated sections are de-hydratedin serial ethanol dilutions and air-dried. Probes are labeled using theMAXIscript in vitro transcription kit (Ambion Inc., Austin, Tex.) anddigoxigenin-UTP (Roche Molecular Biochemicals). Hybridization takesplace overnight at 42° C. Signal detection is carried out with the DIGnucleic acid detection kit (Roche) (67). These test methods determinewhich tissues express a 24-hydroxylated vitamin D compound receptorduring development.

RNA is also extracted from several adult tissues (brain, muscle,intestine, kidney, liver, spleen, skin, testis/ovaries, bone, etc.). TheRNA is reverse-transcribed using the Applied Biosystems High CapacitycDNA Reverse Transcription Kit (Applied Biosystems, Foster City,Calif.). Relative tissue expression of the 24-hydroxylated vitamin Dcompound receptor is quantified using Reverse Transcription-quantitativePCR (RT-qPCR) on the reverse transcribed mRNA from different tissueswith a specific TaqMan (Applied Biosystems) probe. The RT-qPCR reactionis performed on an Applied Biosystems 7500 instrument (AppliedBiosystems) by the comparative ΔC_(t) method and normalized to Gapdh.These experiments determine the tissue distribution of the24-hydroxylated vitamin D compound receptor mRNA expression.

The expression patterns are confirmed using immunochemistry. Since itremains challenging to purify recombinant membrane proteins, thesemethods instead raise anti-peptide antibodies to the 24-hydroxylatedvitamin D compound receptor. Antigenic peptides are identified using theAntigen Profiler™ algorithm(www.openbiosystems.com/antibodies/custom/AntigenProfiler/). Antibodiesto these antigenic peptides are used to probe 24-hydroxylated vitamin Dcompound receptor protein expression in tissues identified through theRT-qPCR assay, as well as in intact and fractured bones. Bones aredissected, are fixed overnight in 4% paraformaldehyde, de-mineralized in0.5M EDTA (68), and are embedded in paraffin for immunohistochemistry on6 μm sections with the anti-24-hydroxylated vitamin D compound receptorantibodies.

Functional immunohistochemistry protocols are developed for murine bonesections (69) using the Retrievagen A antigen retrieval system (BDBioSciences Canada). Briefly, fixed, deparaffinized, rehydrated sectionsare treated with Retrievagen A for 10 minutes at 94° C., are blockedwith the M.O.M. blocking reagent (Vector Laboratories, Burlingame,Calif.), and are incubated with the primary antibody. Detection usesenzyme-conjugated or fluorochrome-conjugated secondary antibodies. Theseexperiments confirm the RNA expression profiling data and identify whichcell type(s) express the 24-hydroxylated vitamin D compound receptor inbone.

Example 4 Characterization of the Signal Transduction Pathway Downstreamfrom the 24-Hydroxylated Vitamin D Compound Receptor

To identify the signal transduction pathway acting downstream of the24-hydroxylated vitamin D compound ligand—24-hydroxylated vitamin Dcompound receptor interaction, the commercial Cignal Finder reportersystem from SABiosciences (Frederick, Md.) is used. This system consistsof sets of vectors that each contain a distinct cis-acting enhancerelement upstream of the luciferase reporter gene. These vectors aretransiently transfected into suitable cells. A given stimulus, such asthe binding of a 24-hydroxylated vitamin D compound to its receptor,initiates a signal transduction cascade that ultimately results in thebinding of a specific transcription factor to its response element. Thisin turn leads to increased expression of the reporter gene, providing aconvenient readout.

COS-7 cells stably transfected with the 24-hydroxylated vitamin Dcompound receptor are seeded in 24-well tissue culture plates (Nunc,Roskilde, Denmark) at a density of 20,000 cells per well in growthmedium (high-glucose DMEM with HEPES, 10% FBS, and 400 μg/ml ofGeneticin). The next day the medium is changed to 0.5 ml growth mediumwithout selection antibiotic. The cells are then transfected with theCignal AARE Reporter vector (SABiosciences, Frederick, Md., USA): 250 ngof DNA is mixed with 50 μl of OptiMEM (Invitrogen, Grand Island, N.Y.,USA) medium and 1.6 μl of Surefect transfection reagent is mixed with 50μl of OptiMEM. After a five minute incubation period, the DNA andtransfection reagent are mixed, further incubated for twenty minutes,and the mixture is deposited on top of the cells. After 24 hours, themedium is changed to serum-free DMEM, and the cells are starvedovernight (16-18 h). Then, different concentrations of 24-hydroxylatedvitamin D compound are added to the cells. Following incubation, cellsare washed with PBS and lysed with lysis buffer. The activity of fireflyand renilla luciferases are measured sequentially on a SiriusLuminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). Thespecific luciferase activity is expressed as the ratio offirefly/renilla luciferase activity. The specific luciferase activity ofeach treatment group is normalized to the specific activity of acontrol, vehicle-treated group. These values are shown in the graphs asrelative luciferase activity.

The Cignal AARE reporter vector detects the pathway that responds to theATF family of transcription factors. This pathway shows specificinduction in response to 24-hydroxylated vitamin D compound treatment ofcells stably transfected with an expression vector for the24-hydroxylated vitamin D compound receptor (FIG. 5). This result isparticularly significant considering the established key roles of theATF4 transcription factor in all aspects of osteoblast biology. Theidentification of the transcription factor involved in mediatingresponses downstream from the 24-hydroxylated vitamin D compoundreceptor allows to screen for vitamin D compounds that have agonistic orantagonistic activity for the receptor.

As a first step to confirm the binding of the transcription factor toits cognate response element upon ligand-receptor interaction, we useElectrophoretic Mobility Shift Assays (EMSAs). Oligonucleotidescorresponding to the ATF4 consensus binding site are synthesized andtested for binding using nuclear extracts in EMSAs. Nuclear extracts areprepared as described previously (73) using the technique of Andrews andFaller (75). Ten micrograms (10 μg) of nuclear proteins are incubatedfor 30 min at 4° C. in 20 μl of binding buffer (100 mM Tris-HCl, pH 7.5,20 mM MgCl₂, 500 mM NaCl, 2% NP-40, 10 mM DTT, 10 mM EDTA, 100 ng ofpolydI-dC, 30% Ficoll). Labeled probe (5000 dpm) is then added to thebinding reaction mixture. The binding reactions are size-fractionated onnon-denaturing 6% polyacrylamide gels, then the gel is dried andautoradiographed. Binding of ATF4 to the probe is induced by24-hydroxyvitamin D compound treatment of the cells and result in acomplex with reduced electrophoretic mobility (70-75).

In parallel, the pathways operating upstream of ATF4 but downstream fromthe liganded 24-hydroxylated vitamin D compound receptor arecharacterized. These pathways involve protein kinase signaling, such asprotein kinase A, the cAMP-dependent protein kinase. Promega's SignaTECTprotein kinase assay systems (Promega Corporation, Madison, Wis.) areused to characterize specific protein kinase pathways operatingdownstream of the 24-hydroxylated vitamin D compoundligand—24-hydroxylated vitamin D compound receptor interaction. Thissystem overcomes the drawbacks of commonly used kinase assay methodsthat rely on the capture of phosphorylated peptide substrates onphosphocellulose (80). The SignaTECT assay is straightforward andrequires phosphorylation and binding of a biotinylated substrate to abiotin capture membrane. Unincorporated [γ-³²P]ATP is removed by asimple wash procedure. Washing also removes nonbiotinylated proteinsthat have been phosphorylated by other kinases in the sample. The bound,labeled substrate is quantitated by scintillation counting,phosphorimaging analysis or by using autoradiography. SignaTECT ProteinKinase Assay Systems are available for cAMP-Dependent protein kinase,protein kinase C, calcium/calmodulin-dependent protein kinase II (CaMKII), DNA-dependent protein kinase, tyrosine kinases, and cdc2 proteinkinase.

These experiments identify and characterize the signaling pathways thatoperate to amplify the signal downstream of the interaction of the24-hydroxylated vitamin D compound ligand with its receptor. Theestablishment of a cell line with an ATF4 reporter system activated uponbinding of the D metabolite to its receptor is a useful tool to screenfor vitamin D compounds that bind the receptor with increased affinityor specificity, or that display antagonist properties.

Example 5 Characterization of Physiological Role of the 24-HydroxylatedVitamin D Compound Receptor in Fracture Repair

Since mice deficient for Cyp24a1 cannot synthesize any 24-hydroxylatedvitamin D compound ligand and exhibit a delay in callus formation duringfracture healing, mice with a targeted mutation in the 24-hydroxylatedvitamin D compound receptor show a similar phenotype. In the presentExample, a strain of mice with a conventional knockout mutation as wellas a strain allowing cell-type specific inactivation of the24-hydroxylated vitamin D compound receptor gene are established. Thissection first describes the gene targeting strategies, followed by theosteotomy/fracture repair procedure, and finally assays for phenotypeanalysis.

Embryonic stem cells targeted at a 24-hydroxylated vitamin D compoundreceptor locus through gene trapping have been identified within thepublicly available collection of the Texas A&M Institute for GenomicMedicine (TIGM). In this clone, the promoter-less marker/reporter genetrap is inserted into the first intron of the target 24-hydroxylatedvitamin D compound receptor gene. This leads to an incorrect splicing ofthe target gene in which the first exon will be fused to the markersequence to create a marker fusion transcript that can be detected bystaining for β-galactosidase activity. All exons downstream of theinsertion site are not expressed, leading to inactivation of the trappedgene. This targeted ES cell clone is purchased, which considerablyreduces the time required to engineer a conventional knockout strain.The marker fusion transcript allows further refinement in the study ofthe expression pattern of the target 24-hydroxylated vitamin D compoundreceptor gene.

In parallel, a targeting vector based on the Cre/lox technology toachieve cell-type specific inactivation of the target 24-hydroxylatedvitamin D compound receptor gene is engineered. A 129Sv bacterialartificial chromosome (BAC) clone encompassing the 24,25(OH)₂D receptorgene locus is commercially available. Using the technique ofrecombineering (recombination-mediated genetic engineering) (81-83), atargeting vector is constructed in which loxP sites are inserted withinintron 1 and downstream of exon 5. Cre-mediated excision between thoseloxP sites delete 7140 basepairs containing exons 2-5, which essentiallyrepresents the entire coding sequence of the gene. The linearizedtargeting vector is electroporated into R1 ES cells (84) and doubleselection with the aminoglycoside antibiotic G418 and the nucleosideanalog gancyclovir are applied (85). Resistant colonies are picked andexpended into cell lines; these are screened for the presence of thedisrupted 24-hydroxylated vitamin D compound receptor gene by Southernblot analysis after preparation of DNA by the micro-isolation techniqueof Laird et al (86).

ES cell clones carrying the gene-trapped allele or the floxed allele areexpanded and then injected into C57BL/6 embryos at the blastocyst stage.Chimeric animals born from these injections are identified on the basisof chimeric coat color (agouti patches on a black background). Chimericmales are bred to C57BL/6 females and germ line transmission assessed bythe presence of the agouti coat color in the resulting F1 progeny.Animals showing germ line transmission are genotyped by Southern blotanalysis of tail DNA (86, 87) and heterozygotes for the conventionalknockout or the foxed 24-hydroxylated vitamin D compound receptor alleleare mated inter se to produce animals of all three possible genotypes(+/+, +/− and −/−; or +/+, +/fl, and fl/fl) (33, 67, 88, 89).

The targeted 24-hydroxylated vitamin D compound receptor foxed mice arebred to the Col1-Cre (90) or Col2-Cre (67) to achieve osteoblast- orchondrocyte-specific inactivation of the receptor gene, respectively.This is performed through the following crosses: first, the Cretransgene are bred into the floxed strain (Col1-Cre×fl/fl orCol2-Cre×fl/fl) to obtain mice carrying the Cre transgene and one foxedallele (genotype: Col1-Cre; receptor^(+/fl) or Col2-Cre;receptor^(+/fl)). These mice are mated to homozygote foxed mice togenerate mice with both alleles inactivated in osteoblasts (genotype:Col1-Cre; receptor^(fl/fl)) or chondrocytes (genotype: Col2-Cre;receptor^(fl/fl)). All mice are genotyped through a combination of PCRand Southern blot analysis of tail DNA (86, 87).

Adult wild-type and mutant mice are subjected to a stabilized,transverse mid-diaphysial fracture of the tibia or femur. A currentprotocol makes use of the distraction osteogenesis mouse model (91).This device is a small scale version of the Ilizarov distraction deviceused in orthopaedic patients (92). The custom-designed circular externalfixators consist of two aluminum circular rings held concentrically bytwo stainless-steel threaded rods. Pins for transfixing the bone (0.25mm) are attached to the frame with hexagonal bolts. Under steriletechniques in the procedures room, the proximal metaphysis of the tibiaof anesthetized animals (knockout mutants, tissue-specific deletionmutants, and control littermates) is transfixed with pins drivenpercutaneously with the help of a hand-held variable-speed drill. Thepins are perpendicular to the long axis of the tibia and cross at a 90degrees intersect. Two pins are used to transfix the bone proximally anddistally. The pins are secured to the rings by the hexagonal bolts withthe tibias centered within the frame. A longitudinal incision followedby muscle dissection expose the tibia and a transverse osteotomy isperformed between the two rings. The incision is closed with suturesthat are removed on day 7 (93).

Another protocol to generate a reproducible, aligned fracture is therodded model of immobilized fracture based on the technique described byBonnarens and Einhorn (94). Briefly, closed, transverse, middiaphysealfractures of the femur are generated using an upgrade of the bluntguillotine instrument originally designed for rats (94). Fracturestabilization by intramedullary fixation is carried out using the styletof a 25G spinal needle. The knee joint is flexed and incisions areperformed at the level of the patellar ligament. The ligament isdislocated laterally to expose the femoral condyles. A 26G needle isused to make a hole at the head of the femur through which a 25G spinalneedle is inserted. The needle is then cut and the rodded femur isfractured with the blunt guillotine. After the wounds are closed, aradiograph is taken to confirm the pin placement and the fracture.Animals are permitted full weight-bearing and unrestricted activityafter awakening from anesthesia.

Adult, same gender mice of 4-5 months of age are used with a minimum of5 animals per group. Cohorts are assigned to collect samples forhistology/histomorphometry, while others are assigned to mRNA isolationfor Real Time reverse-transcription PCR. A final cohort is assigned forbiomechanical testing at 21 days post-osteotomy. Blood is collected fromall animals at sacrifice to measure calcemia, phosphatemia, and vitaminD metabolite levels. The fractured legs are dissected at intervalsfollowing surgery (3, 7, 14, 21 days) and are fixed overnight in 4%paraformaldehyde. The bones from the day 7, 14, and 21 cohorts are firstto be analyzed by micro-CT to evaluate bone formation. Then, the fixedlong bones are embedded in methylmethacrylate. Sections of 6 μm aredeplastified and stained by Goldner (68) for comparative histology.Quantitative histomorphometry is performed as described previously (73,95, 96) using the BioQuant Osteo histomorphometry system.

Callus samples isolated at the same intervals (3, 7, 14, 21 dayspost-fracture) from control and mutant mice are also used for mRNAextraction. The mRNA is reverse-transcribed and Real Time PCR isperformed using TaqMan probes for chondrocyte (Sox9, collagen type II,collagen type X, Indian Hedgehog, Hypoxia Inducible Factor-1a) orosteoblast differentiation markers (Osx, Run×2, ATF4, type I collagen,bone sialoprotein, osteocalcin). Additional markers are also tested,including (but not restricted to) VEGF, MMP-9, MMP-13, cyp24a1, cyp27b1,and VDR.

The biomechanical properties of the repaired bones are tested at 21 dayspost fracture and compared between genotypes. For biomechanicalanalysis, bones are collected in normal saline solution and mounted in amodified Instron™ three point bending test apparatus (73, 95, 96).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. For example, but not by way of limitation,the methods described herein for identifying 24-hydroxylated vitamin Dcompounds beneficial for fracture healing that employ animal models areequally indicative of utility in human subjects.

Patents, patent applications, publications, procedures, and the like arecited throughout this application, the disclosures of which areincorporated herein by reference in their entireties.

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What is claimed is:
 1. A method of identifying a compound capable ofbinding to a 24-hydroxylated vitamin D compound receptor, the methodcomprising: (a) contacting a 24-hydroxylated vitamin D compound receptorwith a candidate compound; and (b) determining whether the candidatecompound binds to the 24-hydroxylated vitamin D compound receptor. 2.The method of claim 1, wherein the 24-hydroxylated vitamin D compoundreceptor comprises an amino acid sequence shown in SEQ ID NO: 1 or asequence having at least 90% sequence identity thereto.
 3. The method ofclaim 1, wherein the candidate compound is exposed to a cell expressinga 24-hydroxylated vitamin D compound receptor.
 4. The method of claim 3,wherein binding is detected by measuring a downstream signaltransduction output.
 5. The method of claim 4, wherein binding isdetected by measuring activation of a member of the ATF family oftranscription factors.
 6. The method of claim 5, wherein binding isdetected by measuring activation of ATF4.
 7. The method of claim 4,wherein binding is detected by measuring activation of a protein kinasecapable of directly or indirectly activating a member of the ATF familyof transcription factors.
 8. The method of claim 7, wherein binding isdetected by measuring activation of protein kinase A.